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. 2012 Jun 29;13(7):645-52.
doi: 10.1038/embor.2012.72.

Pds5 promotes cohesin acetylation and stable cohesin-chromosome interaction

Sabine Vaur  1 , Amélie Feytout, Stéphanie Vazquez, Jean-Paul Javerzat
Affiliations

Pds5 promotes cohesin acetylation and stable cohesin-chromosome interaction

Sabine Vaur et al. EMBO Rep. .

Abstract

Pds5 and Wpl1 act as anti-establishment factors preventing sister-chromatid cohesion until counteracted in S-phase by the cohesin acetyl-transferase Eso1. However, Pds5 is also required to maintain sister-chromatid cohesion in G2. Here, we show that Pds5 is essential for cohesin acetylation by Eso1 and ensures the maintenance of cohesion by promoting a stable cohesin interaction with replicated chromosomes. The latter requires Eso1 only in the presence of Wapl, indicating that cohesin stabilization relies on Eso1 only to neutralize the anti-establishment activity. We suggest that Eso1 requires Pds5 to counteract anti-establishment. This allows both cohesion establishment and Pds5-dependent stable cohesin binding to chromosomes.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Pds5 is required for Psm3K106 acetylation. (A) GFP-tagged Psm3 was immunopurified from cycling cells and probed with anti-Psm3K106Ac antibodies. (B) Cells were arrested in G1 by nitrogen starvation and released into rich medium. Progression into S-phase was monitored by measuring DNA content by flow cytometry. Total protein extracts were probed with anti-Psm3K106Ac antibodies and the total amount of Psm3 with anti-Psm3 antibodies. (C) DNA content analysis of cycling cells at 30 °C and after 4-h incubation with hydroxyurea. Total protein extracts were probed with Psm3K106Ac and Psm3 antibodies. The short exposure time shows a slight increase in Psm3 acetylation in the clr6-1 mutant, as reported [19]. The longer exposure (15 min) shows that Psm3 acetylation remains undetectable in a pds5Δ background. GFP, green fluorescent protein; Hu, hydroxyurea; IP, immunoprecipitation; Wt, wild type.
Figure 2
Figure 2
Examination of Rad21 binding to chromosomes in pds5Δ cells upon prolonged G2 arrest. Cycling cells were shifted to 37 °C. The cdc25-22 mutation blocks entry into mitosis and keep cells in G2. (A) Images of Rad21-PK immunofluorescence on nuclear spreads from cells collected before and after 3 h at 37 °C. Chromatin was counterstained with DAPI. Bar=2 μm. (B) Quantification of Rad21 bound to chromosomes. Fluorescence intensity was measured for 50–100 nuclei per sample. Error bar=95% confidence interval of the mean with α=0.05. (C) Cell cycle staging showing that cells were predominantly in G2 before the temperature shift (one nucleus and 2C DNA content) and remained in G2 throughout the experiment. (D) ChiP assay showing the amount of Rad21-PK bound at the indicated loci, shortly (15 min) and 3 h after the 37 °C temperature shift. Error bar=s.d. from four ChiPs. (E) Cytological analysis of cells undergoing their first mitosis upon release from the cdc25-22 arrest. After 3 h at 37 °C, cells were shifted back to 25 °C and fixed 1 h later when most cells were in anaphase. The mitotic spindles were stained by indirect immunofluorescence against tubulin (green) and DNA was stained with DAPI (red, pseudocolour). Bar=5 μm. The frequency of abnormal anaphases was determined from the examination of 80–100 anaphases per sample. Abnormal anaphases were defined as cells with a spindle length>5 μm displaying DAPI-stained material that had not reached the spindle poles. (F) Cell survival was determined by plating cells back to 25 °C and scoring the number of colony-forming units. Error bar=s.d. from four (control) and five (pds5Δ) experiments. ChIP, chromatin immunoprecipitation; DAPI, 4,6-diamidino-2-phenylindole.
Figure 3
Figure 3
Pds5 is required for the stable mode of cohesin interaction with chromosomes. (A) Kinetics of Rad21 dissociation from G2 chromosomes upon inactivation of the cohesin loader. Cycling cells were shifted to 37 °C to shut off cohesin loading (mis4-367) while keeping cells in G2 (cdc25-22). Chromatin-bound Rad21 was measured at the indicated time points by nuclear spreads (50–100 nuclei per sample). Error bar=95% confidence interval of the mean with α=0.05. (B) Cell survival over time. Error bar=s.d. from three experiments. (C) Cell cycle staging showing that cells were predominantly in G2 before the temperature shift and remained in G2 throughout the experiment. (D) ChiP assay showing the amount of Rad21-PK bound at the indicated loci, shortly (15 min) and 3 h after the 37 °C temperature shift. Error bar=s.d. from three ChiPs. ChIP, chromatin immunoprecipitation.
Figure 4
Figure 4
The absence of a stable cohesin fraction in pds5Δ is not rescued by wpl1 deletion. Cycling cells were shifted to 37 °C to shut off cohesin loading (mis4-367) while keeping cells in G2 (cdc25-22). (A) Kinetics of Rad21 dissociation from chromatin by nuclear spreads. Fluorescence intensity was measured for 50–100 nuclei per sample. Error bar=95% confidence interval of the mean with α=0.05. (B) Cell survival over time. Error bar=s.d. from three experiments. (C) Cell cycle staging before and after the temperature shift. (D) ChiP assay showing the amount of Rad21-PK bound at the indicated loci, shortly (15 min) and 3 h after the 37 °C temperature shift. Error bar=s.d. from four ChiPs. (E) Cytological analysis of cells undergoing their first mitosis upon release from the cdc25-22 arrest. Green: tubulin, red: DAPI. Bar=5 μm. The frequency of abnormal anaphases was determined as in Fig 2 from the examination of 80–100 anaphases per sample. ChIP, chromatin immunoprecipitation; DAPI, 4,6-diamidino-2-phenylindole.
Figure 5
Figure 5
Pds5’s function in cohesion maintenance remains essential in the absence of both Eso1 and Wpl1. (A) Kinetics of Rad21 dissociation from G2 chromosomes upon inactivation of the cohesin loader. Cycling cells were shifted to 37 °C to shut off cohesin loading (mis4-367) while keeping cells in G2 (cdc25-22). Chromatin-bound Rad21 was measured by nuclear spreads (50–100 nuclei per sample). Error bar=95% confidence interval of the mean with α=0.05. (B) Cell survival over time. Error bar=s.d. from three experiments. (C) Cell cycle staging showing that cells were predominantly in G2 before the temperature shift and remained in G2 throughout the experiment. (D) ChiP assay showing the amount of Rad21-PK bound at the indicated loci, shortly (15 min) and 3 h after the 37 °C temperature shift. Error bar=s.d. from four ChiPs. (E) Cytological analysis of cells undergoing their first mitosis upon release from the cdc25-22 arrest. Green: tubulin, red: DAPI. Bar=5 μm. The frequency of abnormal anaphases was determined as in Fig 2 from the examination of 80–100 anaphases per sample. ChIP, chromatin immunoprecipitation; DAPI, 4,6-diamidino-2-phenylindole.
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